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Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Nature Biotechnology volume 23, pages 94–101 (2005)Cite this article

Abstract

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

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Figure 1: Characteristics of the immunoaffinity profiling strategy.
Figure 2: Immunoaffinity profiling of NIH 3T3 cells expressing oncogenic Src Y527F.
Figure 3: Distribution of amino acid residues surrounding phosphotyrosine among the phosphopeptides identified from three distinct cell types.

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Acknowledgements

We are grateful to Jeffrey Knott, Jason Reynolds and Matthew Prokop for providing synthetic peptides, Sandra Schieferl for the 3T3-Src cell line and David Mason for advice on the use of ALCL cell lines. We thank Ross M. Tomaino, Scott A. Gerber and Steven P. Gygi for helping us collect tandem mass spectra in the early stages of this project, and Daniel Schwartz, Sean A. Beausoleil, Rob Duarte and Steven P. Gygi for producing heat maps for the phosphotyrosine peptides we identified. This work was supported by a Small Business Innovation Research phase I grant 1R43CA101106 of the Innovative Molecular Analysis Technologies program from the National Cancer Institute (J.R.).

Author information

Author notes

  1. Hui Zhang

    Present address: Institute for Systems Biology, 1441 N. 34th St., Seattle, Washington, 98103, USA

  2. Xiang-Ming Zha

    Present address: Department of Internal Medicine, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, USA

Authors and Affiliations

  1. Cell Signaling Technology Inc., 166B Cummings Center, Beverly, 01915, Massachusetts, USA

    John Rush, Albrecht Moritz, Kimberly A Lee, Ailan Guo, Valerie L Goss, Erik J Spek, Hui Zhang, Xiang-Ming Zha, Roberto D Polakiewicz & Michael J Comb

  2. Institute for Systems Biology, 1441 N. 34th St., Seattle, Washington, 98103, USA

    Xiang-Ming Zha

  3. Department of Internal Medicine, Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, USA

    Hui Zhang

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Supplementary information

Supplementary Fig. 1

The reference library-searching strategy. (PDF 269 kb)

Supplementary Table 1

Phosphotyrosine peptides from signaling proteins found in pervanadate-treated Jurkat cells (PDF 52 kb)

Supplementary Table 2

Phosphotyrosine peptides from Jurkat-pervanadate-trypsin/P-Tyr-100 (PDF 70 kb)

Supplementary Table 3

Phosphotyrosine peptides found in Jurkat-pervanadate-trypsin 15–25% acetonitrile fraction/P-Tyr-100 during reproducibility studies (PDF 55 kb)

Supplementary Table 4

Phosphotyrosine peptides from signaling proteins found in 3T3-Src (PDF 54 kb)

Supplementary Table 5

Phosphotyrosine peptides from 3T3-Src-trypsin/P-Tyr-100 (PDF 70 kb)

Supplementary Table 6

Comparison of non-redundant phosphotyrosine peptides from anaplastic large cell lymphoma cell lines, Karpas 299 or SU-DHL-1-trypsin/P-Tyr-100 (PDF 65 kb)

Supplementary Table 7

Phosphotyrosine peptides from anaplastic large cell lymphoma cell lines, Karpas 299 or SU-DHL-1-trypsin/P-Tyr-100 (PDF 106 kb)

Supplementary Table 8

Phosphotyrosine peptides from SU-DHL-1-trypsin or chymotrypsin or endoproteinase GluC or elastase/P-Tyr-100 (PDF 78 kb)

Supplementary Table 9

Number of above-threshold peptide assignments from forward and reverse databases (PDF 33 kb)

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Rush, J., Moritz, A., Lee, K. et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23, 94–101 (2005). https://doi.org/10.1038/nbt1046

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